Intestinal acetate and butyrate availability is associated with glucose metabolism in healthy individuals

Summary Animal studies suggest that short-chain fatty acids acetate and butyrate are key players in the gut-brain axis and may affect insulin sensitivity. We investigated the association of intestinal acetate and butyrate availability (measured by butyryl-coenzyme A transferase (ButCoA) gene amount) with insulin sensitivity and secretion in healthy subjects from the HELIUS cohort study from the highest 15% (N = 30) and the lowest 15% (N = 30) intestinal ButCoA gene amount. The groups did not differ in insulin sensitivity or secretion. However, the high ButCoA group showed lower glucose and insulin peaks during the first 60 min after a meal and a higher nadir during the second 60 min (p < 0.01), suggesting delayed glucose adsorption from the small intestine. Our data suggest that chronically increased acetate and butyrate availability may improve glucose metabolism by delaying gastric emptying and intestinal adsorption. Future studies should further investigate the effect of acetate and butyrate interventions.


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OPEN ACCESS availability (estimated as the fecal concentration of the butyryl-coenzyme A (CoA)-transferase (ButCoA) gene 14 ) from the large HELIUS cohort. 15The link between ButCoA and intestinal SCFA turnover is supported by multiple lines of evidence.First, the study of Diez-Gonzalez et al. 14 showing a clear positive relation between ButCoA activity and acetate in the butyrate producer B. fibrisolvens.Second, many abundant gut butyrogens like Faecalibacterium, Anaerostipes, and Anaerobutyricum spp.incorporate exogenous acetate to produce butyrate via ButCoA in an ATP generating reaction, supporting the relation between ButCoA abundance and acetate and the direct relation of ButCoA abundance and butyrate. 16,17Third, early textbooks that may have not received great attention already described the role of ButCoA in Clostridium subspecies. 18Glucose and insulin responses during a standardized mixed meal test (SMMT) were measured, as well as ghrelin and GLP-1.In addition, we determined neural activity in reward and satiety related areas in response to visual food cues and the anticipation and receipt of palatable food, by means of functional magnetic resonance imaging.

Clinical characteristics
A total of 30 subjects with high ButCoA gene abundance (mean 367.07%G 205.97% ButCoA copies per 16S rRNA gene copy) and 30 subjects with low ButCoA gene abundance (mean 7.17% G 7.93% ButCoA copies per 16S rRNA gene copy) were included in this study.The ButCoA gene abundance was used as a proxy for high or low intestinal ButCoA levels over a longer period of time. 14Concentrations of the ButCoA gene can be used as a measure for intestinal acetate and butyrate availability. 19High ButCoA gene abundances correspond to high intestinal acetate and butyrate concentrations. 16,17Clinical and biochemical characteristics of the subjects per group (Table 1) showed no differences in baseline demographics between the two groups.No important differences in medication use were found between the groups.Subjects used the following medication: proton pump inhibitors (1 subject in the low and 3 in the high ButCoA group), oral anticoagulants (1 subject in the low and 2 in the high acetate/butyrate group), oral contraceptives (1 subject in the low and 2 in the high ButCoA group), antihypertensive medication (3 subjects in the low and 1 in the high ButCoA group), and statins (1 subject in the high ButCoA group).There were also no differences between groups in fecal SCFA levels, VAS hunger score, or mean daily caloric intake or consumption of carbohydrates, fat, protein, or dietary fiber (Table 1).GLP-1 differences were tested by comparing the 2-h area under the curve (AUC) (mean 905.90 and 954.05 mmol/L x min in the low and high ButCoA group, respectively, p = 0.455).Ghrelin 1.5-h AUC did not significantly differ between groups (mean 845.7 and 864.8 pmol/L x min, p = 0.633).Since absolutely no trend toward significance was observed, and because of logistic and financial limitations, the ghrelin assay was not repeated for the entire study population.

Glucose and insulin responses differed between groups
Fasting and postprandial glucose and insulin levels are depicted in Figure 1.There was no difference between the low and the high ButCoA group in insulin sensitivity estimated by Matsuda index, oral glucose insulin sensitivity (OGIS) index, or homeostatic model assessment for insulin resistance (HOMA-IR).Insulin secretion assessed by insulinogenic index did not differ between groups.Data are depicted in Table 2.
However, the two groups differed significantly in glucose and insulin curves over time during the 2-h period after the meal.The high ButCoA group showed lower plasma glucose and insulin values during the first 60 minutes after the SMMT, but higher levels during the second 60 minutes compared to the low group (Figure 1) (linear mixed model p < 0.01).This difference in postprandial glucose and insulin levels between high and low acetate producers persisted when analyzing only the overweight participants (BMI >25.0) (p = 0.045 and p = 0.0005, respectively), but was not observed when analyzing only the lean participants (p = 0.184 and p = 0.793).
Finally, the difference persisted upon exclusion of participants using medication affecting gastrointestinal motility (4 in the low and 4 in the high ButCoA group, respectively) (p = 0.01 and p = 0.006, respectively).

No differences between groups in CNS responses while viewing food pictures
On whole brain level, we found a significant main effect for contrast 1 (food vs. non-food images) in the right insula and left orbitofrontal cortex (OFC) regions across both groups (Figure 2).Additionally, we found a main effect for contrast 2 (high-caloric vs. non-food images) in the bilateral insula and OFC (Table S1; Figure S2).However, no significant between-group differences were found in the predetermined ROIs for the viewing of food pictures.

Lower left putamen activation in anticipation of food receipt in high ButCoA group compared to low ButCoA group
No main effects were observed on whole brain level across groups.When performing group-comparisons in the selected ROIs, during the anticipation of chocolate milk vs. tasteless solution, the activity in the left putamen tended to be lower in the high acetate group, compared to the low ButCoA group (respectively -0.035[-0.069-0.013]% and 0.033[-0.016-0.074]% blood-oxygen-level-dependent (BOLD) signal change, Bonferroni adjusted p value = 0.088) (Figure 2E).Furthermore, no significant left putamen activity was found in any of the groups when compared to 0% BOLD signal change in a one-sample t test.

Fecal microbiota composition in relation to intestinal acetate availability
Using univariate analyses, no specific differences in amplicon sequence variants (ASVs) were observed between the low and the high intestinal ButCoA group, after false discovery rate correction for multiple comparisons.When applying an XGBoost machine learning classification model predicting whether a subject belonged to the low or high group, an AUC of 0.667 was obtained, only remotely higher than the permuted model, which had an AUC of 0.625, indicating a low predictive power of the actual model.Nevertheless, the true model median compared to all permuted outcome iterations differed significantly (p = 6.29eÀ07).Furthermore, no corresponding predictor ASVs were found between the real and the permuted model.The top ASVs with the highest feature importance for this model signified: Agathobacter spp.(previously known as the Eubacterium rectale group), Lachnospiraceae spp., Anaerostipes hadrus, and Dialister invisus (see Figures S1  and S4 for complete results).Of the top 10 predictor ASVs, the following significantly correlated to intestinal ButCoA concentrations: Agathobacter spp.and Lachnospiraceae spp.(Figure 4).

DISCUSSION
Here, we report a study in humans to investigate the association of intestinal acetate and butyrate availability with insulin sensitivity and insulin secretion as well as neural activity in reward and satiety-related areas.Insulin sensitivity and insulin secretion were similar, but glucose and insulin responses upon a standard high fiber meal were significantly lower in the high ButCoA group during the first hour and higher during the second postprandial hour.Interventional studies in animals suggested that oral or colonic acetate or butyrate administration may improve insulin sensitivity and induce satiety. 6,7This was supported by a subgroup analysis of a preliminary study using oral butyrate supplementation. 12However, we did not find a relationship between intestinal acetate and butyrate availability (as determined by fecal ButCoA gene abundance) with insulin sensitivity or secretion in humans.Nevertheless, the high ButCoA group showed significantly different glucose and insulin responses upon a standard high fiber meal (p < 0.01).The high ButCoA group had lower glucose and insulin levels in the first hour, yet higher levels in the second hour after the mixed meal.In other words, both glucose and insulin had lower excursions in individuals with high intestinal acetate and butyrate availability.These data suggest a delayed glucose adsorption from the small intestine.Our results are in line with a preliminary study investigating the effect of vinegar on postprandial insulin and glucose in healthy humans. 20Vinegar consists largely of acetate and has a significant effect on postprandial glucose and insulin response with lower postprandial glycemic and insulinogenic responses in the first hour, but higher values in the second hour.A potential explanation could be the effects of intestinal acetate (as the conjugate base of acetic acid) binding to FFAR2/3 receptors on phasic and tonic gut motility.Additionally, higher SCFA availability in the upper gastrointestinal tract could exert an indirect effect on gastric emptying by stimulating satietogenic hormone secretion such as PYY and GLP-1, which also inhibit gastric motility. 21Delayed gastric emptying induced by GLP-1 receptor agonists 19 resulted in postprandial glucose and insulin responses that are similar to those observed in our study.
We also observed that anticipating the receipt of chocolate milk vs. tasteless solution invoked a lower CNS response in the left putamen in the high ButCoA group compared to the low group, but this did not remain significant after correction for multiple comparisons.We also did not find a main effect for the anticipation of chocolate milk within the putamen or elsewhere in the brain within either group, suggesting that the task did not elicit the desired effect across groups.However, as the main effect includes both groups, it is also conceivable that the contrast elicits opposite effects in two subgroups, or a blunted effect in one of the groups, such that a main effect is not significant.Previous studies demonstrated that obesity is associated with higher responsiveness to palatable food images 22 or cues that predict palatable food receipt. 23The putamen is part of the basal ganglia and is implicated in reward processing and conditioning and has previously been found to play a complex integrating role in food-related reward signals to behavior. 24urrent research indicates that gut microbial-derived SCFAs might be able to affect brain physiology, both through direct and indirect interactions.Firstly, acetate and butyrate bind to FFAR2/3 receptors on enteric nerve cells, hereby inducing secretion of anorectic hormones such as GLP-1 and PYY. 25 Furthermore, some evidence suggests acetate acts directly on hypothalamic centers of appetite control, but can also suppress appetite via the vagal-brainstem-hypothalamic pathway. 26In our current study, we did not administer exogenous (labeled) acetate and therefore we cannot define the exact mechanism of action.However, several studies have shown that higher peripheral levels of acetate correlate with higher CNS concentrations. 5,10For butyrate, central effects have not been shown in humans and are less plausible, since it reaches the periphery in considerably lower concentrations and there is limited evidence for it to cross the blood-brain barrier in significant amounts.However, indirect central effects of butyrate may be plausible.
Our results indicate that both glucose and insulin in individuals with high long-term intestinal acetate and butyrate availability had lower rates of change compared to the low group.Contrary to previous animal studies, 5,27 we did not observe any relation between intestinal SCFA turnover and insulin sensitivity or secretion.However, our findings suggest a delay in gastric emptying or intestinal transit time.Prospective cohort studies should investigate the association of ButCoA gene amount with gastric emptying, intestinal absorption, and glucose metabolism.Future studies should also investigate whether acute or long-term systemic acetate and butyrate administration in humans affects glucometabolic health.

Limitations of the study
First, fecal bacterial species abundancy and SCFA availability may only partially correlate with those in the small intestine, 28 as we did not collect duodenal biopsies due to the invasive nature of this procedure.However, most of the SCFA adsorption takes place in the colon, of which the microbiota composition does correlate well to fecal matter. 14Second, despite the eminent difference in ButCoA levels, we did not observe any differences in fecal SCFA levels between the groups.It has to be noted, though, that these are known to be volatile, highly varying within subjects over time, and correspond poorly to intestinal adsorption rate. 28We therefore selected ButCoA abundance as a more stable measure of intestinal fermenting activity.Third, it could be suggested that the differences between the groups may be explained by confounding factors.Nevertheless, potential confounding factors including age, sex, BMI, medication use, resting metabolic rate, and energy intake did not differ between the two groups, suggesting that the metabolic differences between groups are in fact driven by intestinal metabolic variability, in particular acetate and butyrate availability.Fourth, it is currently not clear through which mechanism of action the SCFA exerts their effect.Gastric emptying has not been measured in our study.Future studies should therefore also include measures of gastric emptying when assessing the effects of SCFA.

Figure 1 .
Figure 1.Glucose and insulin excursions upon mixed meal test (A) Glucose excursions upon mixed meal test.Glucose responses are depicted as mean G S.E.M. Blue line shows the high ButCoA group, red line shows the low ButCoA group.There was no difference in plasma glucose level at baseline between the low and the high ButCoA group (5.13 G 0.572 mmol/L vs. 5.22 G 0.487 mmol/L).(B) Insulin excursions upon mixed meal test.Insulin responses are depicted as mean G S.E.M. Blue line shows the high ButCoA group, red line shows the low ButCoA group.There was no difference in plasma insulin level at baseline between the low and the high ButCoA group (28.10 G 15.24 pmol/L vs. 33.93G 29.32 pmol/L).mmol/L = millimole per liter; SMMT = standardized mixed meal test.

Figure 2 .
Figure 2. fMRI paradigm and outcome (A) Example of timing of picture blocks presentation during the food picture fMRI paradigm.(B) Timing of cue presentation during the chocolate milk fMRI paradigm in unpaired trials.(C) Timing of cue presentation and stimuli delivery during the chocolate milk fMRI paradigm in paired trials.(D) Anatomical mask of left putamen.Images were derived from the Harvard-Oxford subcortical atlas.Probability masks were downloaded and changed into binary masks using the fslmaths command, all images were thresholded at 60%. (E) Group differences in percent response change of BOLD signal upon anticipation of the receipt of chocolate milk vs. tasteless solution between high and low ButCoA group upon exposure to palatable food stimuli with error bars representing standard error.CS = conditioned stimulus; CM = chocolate milk; ISI = interstimulus interval; ITI = inter-trial interval; BOLD = blood-oxygen-level-dependent.

with
Benjamini-Hochberg corrected p-values for multiple comparisons.Relative importance of the top 10 predictor ASVs is depicted in Figure S4.Here, relative importance of the top predictor ASV is set to 100%.Relative bacterial abundance of the top 10 predictor ASVs is depicted in Figure S1.

Table 1 .
Baseline characteristics for the high and low ButCoA group (N = 60) Tested with unpaired t test or Mann-Whitney U test, based on Gaussian distribution.Pearson chi-squared test had been performed to test for differences in sex and medication use between both groups.Numerical values are expressed as means G standard deviations or median [IQR] depending on Gaussian distribution.No differences in characteristics were found between the groups.ButCoA = Butyryl-coenzyme A transferase; BMI = body mass index; bpm = beats per minute; VAS = visual analog scale, SMMT = standardized mixed meal test; fMRI = functional magnetic resonance imaging.

Table 2 .
Differences between the high and low ButCoA group in insulin sensitivity, insulin secretion, and incremental area under the curve (iAUC) of glucose and insulin and the respective ratios Values are expressed as means G standard deviations or medians with interquartile range; differences were assessed using Mann-Whitney U test.No differences were found between both groups.